Abstract

Microorganisms play a fundamental role in the cycling of nutrients and energy on our planet. A common strategy for many microorganisms mediating biogeochemical cycles in anoxic environments is syntrophy, frequently necessitating close spatial proximity between microbial partners. We are only now beginning to fully appreciate the diversity and pervasiveness of microbial partnerships in nature, the majority of which cannot be replicated in the laboratory. One notable example of such cooperation is the interspecies association between anaerobic methane oxidizing archaea (ANME) and sulfate-reducing bacteria. These consortia are globally distributed in the environment and provide a significant sink for methane by substantially reducing the export of this potent greenhouse gas into the atmosphere. The interdependence of these currently uncultured microbes renders them difficult to study, and our knowledge of their physiological capabilities in nature is limited. Here, we have developed a method to capture select microorganisms directly from the environment, using combined fluorescence in situ hybridization and immunomagnetic cell capture. We used this method to purify syntrophic anaerobic methane oxidizing ANME-2c archaea and physically associated microorganisms directly from deep-sea marine sediment. Metagenomics, PCR, and microscopy of these purified consortia revealed unexpected diversity of associated bacteria, including Betaproteobacteria and a second sulfate-reducing Deltaproteobacterial partner. The detection of nitrogenase genes within the metagenome and subsequent demonstration of (15)N(2) incorporation in the biomass of these methane-oxidizing consortia suggest a possible role in new nitrogen inputs by these syntrophic assemblages.

CARD-FISH of Eel River Basin sediment with probe ANME-2c_760 (green) and a general DNA stain (DAPI, blue), before (a) and after (b) capture with paramagnetic beads (dark red, collage of six images). (Scale bars, 10 μm.)

FISH-SIMS acquired profile of carbon-13 and nitrogen-15 within a layered archaeal-bacterial cell aggregate (Inset) recovered from a sediment incubation amended with a headspace containing 15N-dinitrogen and methane. Epifluorescence image of aggregate is shown in Inset, where archaea are depicted in red and bacteria in green. Both natural abundance δ13C (‰) of the archaeal/bacterial aggregate (open diamonds) and fractional abundance (%) 15N (solid circles), representing the amount of 15N label incorporated into aggregate biomass, are depicted. For δ13C values, analytical precision (1σ) is shown. The x axis represents increasing time of Cs+ ion beam exposure during the analysis from left to right (arbitrary units), where aggregate cell material is progressively sputtered away over the course of the analysis. This results in a cross-section profile of 13C and 15N, where values for the outer cell layers (bacteria) are shown on the left and right most portion of the graph, transitioning through the methane-oxidizing archaeal core in the center.